The Ubp8 deubiquitinase forms a tetrameric complex consisting of Ubp8, Sgf73, Sgf11, and Sus1 subunits. We have structural and biochemical evidence that the zinc finger domains of Sgf11 and Sgf73 serve dual functions of organizing the catalytic core of Ubp8 in an enzymatically competent conformation as well as directing substrate specificity toward ubiquitylated histone H2B (H2B). Using X-ray crystallographic and biochemical approaches, we will dissect, at the molecular and atomic levels, the mechanisms of deubiquitinase (DUB) regulation. Removal of ubiquitin from target proteins is essential for cellular processes as fundamental as gene transcription, cell cycle progression, and cell differentiation. As such, mutations, deficiencies, and manipulation of DUBs have been described in several human diseases, namely cancer, progressive neurodegeneration, and pathogenic virus infection. Specifically, Ubp8 (USP22 in humans) has been reported to be a member of an 11-gene signature of cancer stem cells involved in poor cancer outcome prognosis. Sgf73 (Ataxin-7 in humans) has been implicated in an inherited, progressive spinocerebellar ataxia. By elucidating the architecture and regulatory mechanism of this protein complex, we will provide a framework for future research aimed at manipulation of DUB enzymes. These approaches would include, but would not be limited to pharmacological molecule screening, gene therapy, and directed design of novel compounds to activate or inhibit DUB activity. We propose that by potently and specifically controlling the enzymatic activity of deubiquitinases, the pathologic effects of their misregulation may be alleviated. Using X-ray crystallography, we can determine the exact three-dimensional structure of proteins. This structural knowledge can serve as a model to understand how proteins are activated and inhibited by the cell's natural machinery. With this knowledge in hand, we can make informed decisions about how to test and design therapies to activate or inhibit these proteins. Specifically, our study of the Ubp8 complex will guide our understanding of a class of enzymes, known as deubiquitinases, shown to be involved in virtually all biological processes, with fundamental roles in cancer progression, neurodegeneration, and microbial infection. By understanding their three-dimensional structure, and how the parts of this protein complex contribute to its function as a whole, we hope to guide studies aimed at eliminating ailments caused by misregulation of deubiquitinases. PUBLIC HEALTH RELEVANCE: Using X-ray crystallography, we can determine the exact three-dimensional structure of proteins. This structural knowledge can serve as a model to understand how proteins are activated and inhibited by the cell's natural machinery. With this knowledge in hand, we can make informed decisions about how to test and design therapies to activate or inhibit these proteins. Specifically, our study of the Ubp8 complex will guide our understanding of a class of enzymes, known as deubiquitinases, shown to be involved in virtually all biological processes, with fundamental roles in cancer progression, neurodegeneration, and microbial infection. By understanding the three-dimensional structure of the UBp8 complex, and how the individual parts of this protein complex contribute to its function as a whole, we hope to guide studies to eliminate ailments caused by misregulation of deubiquitinases.